One issue in the field of regenerative medicine is the establishment of techniques for inducing pluripotent stem cells in an undifferentiated state, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), to differentiate efficiently into cells having target functions (see Patent Literature 1 to 3 and Non-patent Literature 1 and 2 below).
For example, there is demand for the establishment of methods for culturing (proliferating) pluripotent stem cells in an undifferentiated state, and then inducing the increased numbers of undifferentiated pluripotent stem cells produced by this culture (proliferation) to differentiate into cells having target functions by easy and efficient methods. For example, Patent Literature 3 below describes a technique for inducing differentiation of cultured iPS cells into myocardial cells. In addition, many methods have been reported for inducing pluripotent stem cells in an undifferentiated state to differentiate into myocardial cells, blood cells, reproductive cells, nerve cells and the like.
Recently, there has been increased demand for the establishment of techniques for efficiently inducing pluripotent stem cells in an undifferentiated state to differentiate into endodermal cells (including cells constituting the endoderm, cells differentiated from the endoderm, and cells constituting endodermally-derived tissues).
Examples of endodermally-derived tissues include the liver, gallbladder, kidneys, lungs, stomach, intestines and the like. When these endodermally-derived tissues are damaged (become dysfunctional for example) by inflammation, injury, cancer, lifestyle-related disease or the like, the patient's quality of life (QOL) is often severely impacted. Therefore, there is demand for the establishment of regenerative medicine therapies as one means of effectively treating such dysfunction of endodermally-derived tissues. For example, there is demand for the establishment of techniques for culturing (proliferating) pluripotent stem cells in an undifferentiated state, inducing the increased numbers of undifferentiated pluripotent stem cells produced by this culture (proliferation) to differentiate into endodermal cells by easy and efficient methods, and then supplying the endodermal cells obtained by such differentiation induction to regenerative medicine.
Of the endodermal cells, particular attention has focused on techniques for efficiently inducing differentiation into hepatocytes. The liver serves a very large number of functions in the living body, including metabolism, detoxification, drug metabolism, excretion, and fluid homeostasis. Moreover, the liver is sometimes called the “silent organ” because liver tissue may produce few subjective symptoms, so that damage (dysfunction) is often fairly advanced when treatment is initiated. Liver transplantation (including hepatocyte transplantation) is effective, but conventional transplantation therapy suffers from problems of donor shortage and organ rejection. Thus, hepatocytes and liver precursor cells capable of being used in regenerative medicine would be useful for the establishment therapies to treat liver damage (disease, dysfunction).
The establishment of techniques for efficiently inducing differentiation of pluripotent stem cells into endodermal cells is also anticipated in a variety of fields apart from regenerative medicine, including fundamental medicine, drug discovery, pharmacology and embryology. For example, endodermal cells that have been induced to differentiate from pluripotent stem cells can be used to conduct in vitro research on pathogenesis and drug development for diseases which have been difficult to study in detail in the past. In particular, techniques of inducing differentiation of hepatocytes from pluripotent stem cells hold promise as a means of resolving the problem of stably supplying uniform cells, which is a problem in toxicity evaluation performed using human primary culture hepatocytes in the process of drug development research. Moreover, techniques of inducing differentiation of pancreatic cells from pluripotent stem cells are of interest from the standpoint of supplying insulin that can be used in diabetes treatment.
However, the methods that have been reported for inducing differentiation of pluripotent stem cells into endodermal cells often involve complex operations (for example, differentiation induction methods involving introduction of multiple exogenous genes) or if the operations are relatively easy (addition of multiple kinds of differentiation-inducing compounds in liquid form to medium for example), differentiation efficiency is low. Moreover, differentiation induction methods involving introduction of exogenous genes present safety issues because the exogenous genes are incorporated into genome DNA, while differentiation induction by addition of differentiation-inducing compounds presents cost issues because large quantities of expensive cytokines and other liquid factors are required. Even when it is possible to induce differentiation of pluripotent stem cells into endodermal cells, moreover, the resulting cells often do not function satisfactorily. There is thus demand for methods of inducing differentiation more easily and with greater differentiation efficiency.